Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific.

Australia and New Guinea contain high levels of endemism and biodiversity, yet there have been few evaluations of population-level genetic diversity in fauna occurring throughout the Australo-Papuan region. Using extensive geographical sampling, we examined and compared the phylogenetic relationships, phylogeography and population structure of Anopheles farauti, An. hinesorum and An. irenicus throughout their ranges in the southwest Pacific using mitochondrial (mtDNA COI) and nuclear (ribosomal protein S9 and ribosomal DNA ITS2) loci. Phylogenetic analyses suggest that the ability to utilize humans as hosts has been lost repeatedly, coincident with independent colonizations of the Solomon Islands. As some of the species under investigation transmit malaria in the region, this is a medically important finding. Maximum likelihood and Bayesian phylogenetic analyses of nuclear loci also showed that the three species are monophyletic. However, putative introgression of An. hinesorum mtDNA onto a nuclear background of An. farauti was evident in populations from Queensland, Torres Strait and southern New Guinea. Haplotype networks and pairwise F(ST) values show that there is significant genetic structure within New Guinea and Australia in both An. farauti and An. hinesorum, consistent with a long-term history of low gene flow among populations.

Malaria vectors of Papua New Guinea.

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO(2) baited light traps and human bait collections). A total of 22,970mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.

The anopheline fauna of Papua New Guinea.

Surveys for anopheline mosquitoes were conducted throughout the mainland of Papua New Guinea from 1992 to 1998 with the aim of mapping the distribution of the anopheline fauna. Larval collections, adult trap, and human landing collections indicated the presence of seven species (other than those belonging to the Anopheles punctulatus group); these were An. bancroftii, An. annulipes, An, karwari, An. longirostris, An. meraukensis, An. novaguinensis, and An. subpictus. The distribution and ecology of these species is discussed.

Environmental factors associated with distribution and range limits of malaria vector Anopheles farauti in Australia.

Ecological factors associated with the narrow coastal distribution of Anopheles farauti Laveran s.s. were investigated using decision tree software and a recently developed software tool that permits analysis of environmental gradients across distributional boundaries. Significant variables identified by these procedures were then used to develop ecological niche models that permitted detailed--and improved--predictions of the species' overall distribution. These methods identified seven climatic factors (four of temperature factors and three atmospheric moisture factors) from among 40 environmental variables related to the range of this species. In addition, the gradient-analysis tool identified elevation as being particularly important. The distributional hypothesis predicted using ecological niche modeling of these factors included all of the record sites from which An. farauti s.s. was collected in northern Australia and successfully reconstructed its narrow limitation to coastal areas. Omission of elevation from analyses resulted in unrealistic predictions of potential distributional areas > 100 km inland, where the species has not been found.

Field evaluation of commercial repellent formulations against mosquitoes (Diptera: Culicidae) in Northern Territory, Australia.

Field trials comparing commercially available repellent formulations containing picaridin (1-piperidinecarboxylate acid, 2-(2-hydroxyethyl)-1-methylpropylester) and deet (N,N-diethyl-3-methylbenzamide) against mosquitoes in Northern Territory, Australia, were conducted. Three repellents were compared: Autan Repel containing 9.3% picaridin, RID containing 10% deet, and Bushman Ultra containing 80% deet in a gel. The predominant mosquito species collected were Culex annulirostris Skuse (63.2%), Ochlerotatus normanensis (Taylor) (19.6%), and Anopheles meraukensis Venhuis (8.6%). Autan Repel provided >95% protection against all mosquitoes for 2 h, RID for 7 h, and Bushman for >8 h. Against Cx. annulirostris, Autan Repel provided >95% protection for 5 h, RID for 7 h, and Bushman for >8 h. The study showed that both deet formulations provided significantly better protection against mosquitoes than picaridin (Autan Repel). All 3 repellents provided good protection against Cx. annulirostris, an important vector of arboviruses in Australia.

Field evaluation of repellent formulations containing deet and picaridin against mosquitoes in Northern Territory, Australia.

Field efficacy of repellent formulations containing picaridin (1-methyl-propyl 2-(2-hydroxyethyl)-1-piperidinecarboxylate) or deet (N,N,-diethyl-3-methylbenzamide) against mosquitoes in Northern Territory, Australia, was evaluated. The following repellent treatments were evaluated: 19.2% picaridin (Autan Repel Army 20), a solution of 20% deet in ethanol, and 35% deet in a gel (Australian Defense Force [ADF]). The predominant mosquito species were Culex annulirostris Skuse (57.8%), Anopheles merankensis Venhuis (15.4%), and Anopheles bancroftii Giles (13.2%). The protection provided by repellents against Anopheles spp. was relatively poor, with 19.2% picaridin and ADF deet providing >95% protection for only 1 h, whereas 20% deet provided <95% protection at 1 h after repellent application. In contrast, the repellents provided good protection against Cx. annulirostris, with 19.2% picaridin providing >95% protection for 5 h and both deet formulations providing >95% protection for 7 h when collections ceased. This study provides additional field data showing tolerance of Anopheles spp. for repellents. The response of field populations of Cx. annulirostris, an important vector of arboviruses in Australia, to repellents containing deet and picaridin is reported for the first time.

Molecular identification of the malaria vectors Anopheles anthropophagus and Anopheles sinensis (Diptera: Culicidae) in central China using polymerase chain reaction and appraisal of their position within the Hyrcanus group.

In central China, Anopheles anthropophagus is considered the primary malaria vector and Anopheles sinensis is a secondary vector. Identification of these two cryptic species would facilitate studies on malaria transmission and the application of control measures. At present, the only reliable morphological markers occur in the egg stage, making this approach impractical for any large scale field studies. In this study, we report on the development of a polymerase chain reaction (PCR)-restriction fragment length polymorphism procedure involving the ribosomal DNA ITS2 region for discrimination of these species. The PCR-amplified product size of the ITS2 was 574 bp for An. anthropophagus and 594 bp for An. sinensis. Diagnostic restriction fragment length polymorphisms appeared with the restriction enzymes RsaI or HinfI. This diagnostic PCR was tested on mosquitoes collected from different locations throughout China. Specimens identified morphologically as An. anthropophagus in the adult and egg stage from one location in Quangdong Province were found to be An. sinensis, while specimens from Liaoning Province, which were variable in their egg morphology, were found to be An. anthropophagus. The presence of An. anthropophagus in Liaoning Province extends the range of this species north to 42 degrees N. The ITS2 spacer sequence was used in a maximum parsimony phylogenetic reconstruction of six members of the Hyrcanus group, two members of the Lesteri subgroup, and one member of the Nigerrimus subgroup, with the resulting molecular groupings at odds with the current morphological groupings.

Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea.

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only <10% of bloodmeals were from birds. The predominant species was Cx. annulirostris (88%), with relatively few (4.4%) bloodmeals obtained from humans. From all 12 locations sampled, the mean proportion of Cx. annulirostris fed on pigs (9.1%) was considerably lower than fed on other animals (90.9%). Highest rates of pig-fed mosquitoes (>30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland, Australia.

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.

Field evaluation of repellent formulations against daytime and nighttime biting mosquitoes in a tropical rainforest in northern Australia.

Field trials to compare repellent formulations containing either picaridin or deet against rainforest mosquitoes in northern Queensland, Australia, were conducted. Three repellents were compared at night: 9.3% picaridin and 19.2% picaridin (Autan Repel and Autan Repel Army 20, respectively, Bayer, Sydney, Australia) and 35% deet in a gel (Australian Defense Force [ADF]). During the day, the following three repellents were compared: 19.2% picaridin, 20% deet in a controlled release formulation (Sawyer Controlled Release Deet), and 33% deet in a polymer formulation (U.S. Army Extended Duration Topical Insect and Arthropod Repellent [EDTIAR]). The predominant mosquito species collected was Verrallina lineata (Taylor), with smaller numbers of Ochlerotatus kochi (Donitz), Anopheles farauti s.s. Laveran, Ochlerotatus notoscriptus (Skuse), and Coquilletidia xanthogaster (Edwards). In nighttime tests, 19.2% picaridin provided >94.7% protection for at least 9 h, and ADF deet provided >95% protection for 7 h. The 9.3% picaridin formulation provided >95% protection for only 2 h, and provided 60% protection at 9 h. In daytime tests, Sawyer 20% deet provided >95% protection for 6 h, and both 19.2% picaridin and U.S. Army EDTIAR provided >95% protection for 8 h. In both nighttime and daytime tests 19.2% picaridin provided similar or better protection than deet formulations.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera: Culicidae) subgroup in Australia and the southwest Pacific.

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Speciation and distribution of the members of the Anopheles punctulatus (Diptera: Culicidae) group in Papua New Guinea.

Mosquito collections were made throughout the mainland of Papua New Guinea to identify the members of the Anopheles punctulatus group present and to determine their distribution. Identification was made using morphology, DNA hybridization, and polymerase chain reaction (PCR)-RFLP analysis. Nine members of the group were identified: An. farauti s.s. Laveran, An. farauti 2, An. koliensis Owen, and An. punctulatus Dönitz, were common and widespread; An. farauti 4 was restricted to the north of the central ranges where it was common; An. farauti 6 was found only in the highlands above 1,000 m; and An. farauti 3, An. sp. near punctulatus and An. clowi Rozeboom & Knight were uncommon and had restricted distributions. Identification of An. koliensis and An. punctulatus using proboscis morphology was found to be unreliable wherever An. farauti 4 occurred. The distribution and dispersal of the members of the An. punctulatus group is discussed in regard to climate, larval habitats, distance from the coast, elevation, and proximity to human habitation.

Field evaluation of repellents containing deet and AI3-37220 against Anopheles koliensis in Papua New Guinea.

The repellent 1-(3-cyclohexen-1-yl-carbonyl)-2-methylpiperidine (AI3-37220) was compared with 2 formulations of diethylmethylbenzamide (deet) for its effectiveness in protecting 4 humans against the bites of Anopheles koliensis mosquitoes at a village in Central Province, Papua New Guinea. A mean of 77.2 +/- 10.5 bites/human/10 min of An. koliensis was collected on ethanol-treated (control) volunteers, a much higher density than most previous studies with Anopheles sp. mosquitoes. The protection provided by all repellents did not last long. Both 25% A13-37220 in ethanol and a formulation containing 35% deet in a gel provided >95% protection for only 2 h. A formulation of 25% deet in ethanol provided only 93% protection 1 h after repellent application and 39% protection 5 h after application.

Ribosomal DNA spacer genotypes of the Anopheles bancroftii group (Diptera: Culicidae) from Australia and Papua New Guinea.

Mosquitoes of the Anopheles bancroftii group collected from Northern Australia and Papua New Guinea (PNG) were investigated for sequence variation within the ribosomal DNA ITS2. Wing fringe morphology originally used to identify members of this group was compared to genotypes identified by restriction fragment length polymorphism analysis (RFLP) and heteroduplex analysis (HDA) of the rDNA ITS2. Members of this group separated into four RFLP genotypes (A, B, C and D) with some genotypes displaying wing fringe polymorphisms. Heteroduplex analysis of the ITS2 within and between populations identified genotype A as containing two geographically separate ITS2 sequences: A1 from the Northern Territory of Australia and A2 from Queensland and the Western Province of PNG. Genotypes B and C and genotypes C and D were found sympatric and appeared to be evolving independently suggesting the possibility of cryptic species. Genotype C contained two ITS2 sequence types within the genome.

Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae), a rarely recorded member of the Anopheles punctulatus group.

Anopheline specimens collected in Papua New Guinea were morphologically identified as the rarely recorded Anopheles clowi Rozeboom & Knight. Amplification of the rDNA ITS2 region of this material revealed a fragment of 750 bp confirming its placement in the Anopheles punctulatus group. This group contains 12 species and includes the major malaria vectors in the islands of the southwest Pacific. Digestion of the ITS2 with the restriction enzyme MspI produced restriction fragment-length polymorphism with bands at 380, 300, and 150 bp, a pattern shared by no other members of this group. Phylogenetic analysis involving the sequencing of a 2 kb region of the rDNA 18S gene indicated that An. clowi was monophyletic and basal to the rest of the group and showed considerable independent evolution from the other members. This is the first record of An. clowi in Papua New Guinea and only the third collection of this species since its discovery in 1945.

Differential ecology of Anopheles punctulatus and three members of the Anopheles farauti complex of mosquitoes on Guadalcanal, Solomon Islands, identified by PCR-RFLP analysis.

From a series of larval collections made across northern Guadalcanal during the dry season, October-November 1997, four members of the Anopheles punctulatus group of mosquitoes (Diptera: Culicidae) were identified using PCR-RFLP analysis. Anopheline larvae were found in 54/57 (95%) of the sites sampled, comprising An. farauti Laveran sensu stricto (32 sites), An. farauti species no. 2 (39 sites), An. farauti no. 7 (36 sites) and An. punctulatus Dönitz (10 sites). Anopheles punctulatus occurred only on the coastal plain, where it was associated with the more transient sites. Anopheles farauti sensu lato was more widespread throughout the survey region, with similar proportions of all three sibling species in both transient and permanent sites. Two members of the An. farauti complex, An. farauti s.s. and species no. 2, were found in brackish water. All breeding sites of An. punctulatus were cohabited by An. farauti s.l., sometimes by all three sibling species. Anopheles farauti s.s. was the only species collected on human bait, with a much higher biting rate early in the evening (57 bites/human/hour at 18.30-20.00 hours) than later (0.8 bites/human/hour at 21.00-24.00 hours).

Subset partitioning of the ribosomal DNA small subunit and its effects on the phylogeny of the Anopheles punctulatus group.

A phylogenetic study, based on maximum parsimony, of ten species in the Anopheles punctulatus group of malaria vectors from the south-west Pacific was performed using structural and similarity-based DNA sequence alignments of the nuclear small ribosomal subunit (SSU = 18S). The structural alignment proved to be more informative than a computer generated similarity-based alignment. Analyses involving the full structural sequence alignment (2169 bp) and the helical regions (1547 bp) resolved a single tree of the same topology, while analyses using the similarity based alignment could not resolve the group. Studies on the three structural domains of the nuclear rDNA SSU identified domain 2 (769 bp) as the only region informative at the sibling-species level and resulted in the same tree as the full structural sequence and helical regions. The main conclusions of these studies were that the An. punctulatus group formed two clades: a Farauti clade containing members displaying an all black scaled proboscis (An. farauti 1-3 and 5-7) and a Punctulatus clade containing members that display some degree of white scaling on the proboscis (An. farauti 4, An. punctulatus and An. species near punctulatus). Anopheles koliensis can display either proboscis morphology and was positioned basal to the Farauti Clade. These results do not fully concord with those derived from the mitochondrial COII gene.

Populations of the south-west Pacific malaria vector Anopheles farauti s.s. revealed by ribosomal DNA transcribed spacer polymorphisms.

Malaria in the south-west Pacific is transmitted by members of the Anopheles punctulatus group which comprises 12 cryptic species with overlapping morphology. The most widely distributed species of the group is Anopheles farauti s.s. (An. farauti 1) found throughout northern Australia, Papua New Guinea, eastern Indonesia, the Solomon Islands and Vanuatu. A study of the population structure of this species using PCR-RFLP analysis on the ribosomal DNA internal transcribed spacer 1 reveals five genotypes which had distinct geographical distributions. Where these distributions overlap, genotype hybrids can be identified. Heteroduplex analysis of the ITS2 region reveals combinations of nonhomogenized ITS2 sequences and subsequently seven identifiable genotypes, reflecting the ITS1 distribution. Sequence analysis of these ITS2 polymorphisms reveals a minimum of 13 ITS2 sequence types present in heterogeneous combinations in individual mosquitoes. It appears that there are different levels of evolution occurring within the ITS1 and ITS2 regions. These data suggest that An. farauti s.s. may contain multiple loci for the rDNA gene family or that the homogenization of these regions is relatively slow and can be used in genetic studies of population distribution and structure.

Systematics of malaria vectors with particular reference to the Anopheles punctulatus group.

The appearance of groups and complexes of closely related cryptic or sibling species in many of the anopheline taxa has impeded studies on malaria transmission and the evaluation of control strategies which have relied on morphological characters to identify the vector species involved. The advantages of morphological identification are low cost, speed and simplicity, which allow large numbers of specimens to be processed rapidly in the field. The need for accurate identification is crucial, as time and money may be wasted in studying and controlling species of no medical importance. Various techniques such as cross-mating, chromosome studies and allozyme analysis have been developed to resolve problems of identifying sibling species, though none, as yet, can match the speed and simplicity afforded by morphology markers. The latest of these identification methods comes from advances that have been made in DNA-based technology. Although costly and requiring fairly sophisticated laboratory support, methods such as DNA probe hybridisation and PCR are the quickest and most user-friendly to date. The use of DNA has other advantages in the study of intraspecific differences and in providing characters for phylogenetic studies. This review looks at the development of DNA-based techniques for taxonomic and systematic studies of anopheline mosquitoes. The Anopheles punctulatus group of the southwest Pacific is featured as an example of how this technology has been applied and how it has progressed.

A phylogenetic study of the Anopheles punctulatus group of malaria vectors comparing rDNA sequence alignments derived from the mitochondrial and nuclear small ribosomal subunits.

A phylogenetic study of the members of the Anopheles punctulatus group was performed using structural and similarity-based DNA sequence alignments of the small ribosomal subunit (SSU) from both the nuclear and the mitochondrial genomes. The mitochondrial SSU gene (12S, approximately 650 bp) proved to be highly restricted by its secondary structure and displayed little informative sequence variation. Consequently, it was considered unsuitable for a phylogenetic study of these closely related mosquito species. A structural alignment of the nuclear ribosomal DNA SSU (18S, approximately 2000 bp) proved to be more informative than similarity-based alignments. Analyses showed the A. punctulatus group to be monophyletic with two major clades; a Farauti clade containing members displaying an all-black-scaled proboscis (A. farauti 1-3 and 5-7) and the Punctulatus clade containing members displaying extensive white scaling on the apical half of the proboscis (A. farauti 4, A. punctulatus, and An. sp. near punctulatus). Anopheles koliensis was positioned basal to the Farauti clade.